Ann Rheum Dis: first published as 10.1136/annrheumdis-2020-219247 on 18 February 2021. Downloaded from Recommendation ‘SMASH’ recommendations for standardised microscopic arthritis scoring of histological sections from inflammatory arthritis animal models Silvia Hayer ,1 Margriet J Vervoordeldonk,2,3 Maria C Denis,4 Marietta Armaka,5 Markus Hoffmann,6 Johan Bäcklund,7 Kutty Selva Nandakumar ,7,8 Birgit Niederreiter,1 Christina Geka,4 Anita Fischer,1,9 Nina Woodworth,10 Stephan Blüml ,1 George Kollias ,5,11 Rikard Holmdahl ,7 Florence Apparailly,12 Marije I Koenders13 Handling editor Thomas Pap ABSTRACT synovial inflammation causing structural joint Animal models for inflammatory arthritides such damage and functional disabilities.1 2 Numerous ► Additional material is published online only. To view, as rheumatoid arthritis (RA) and psoriatic arthritis animal models that closely resemble characteristic please visit the journal online are widely accepted and frequently used to identify features found in patients with arthritis are studied (http:// dx. doi. org/ 10. 1136/ pathological mechanisms and validate novel therapeutic worldwide to identify novel pathologenetic mech- annrheumdis- 2020- 219247). strategies. Unfortunately, many publications reporting anisms or to validate novel therapeutic approaches on these animal studies lack detailed description and (figure 1).3–5 Based on the complexity of the For numbered affiliations see end of article. appropriate assessment of the distinct histopathological disease, it is of particular importance to correctly features of arthritis: joint inflammation, cartilage damage address the effects of therapeutic agents, or other Correspondence to and bone erosion. Therefore, the European consortium interventions, such as gene knock- ins or knock- PhD Silvia Hayer, Department of BeTheCure, consisting of 38 academic and industrial outs on the distinct pathophysiological features Internal Medicine III, Division of partners from 15 countries, set as goal to standardise including synovial inflammation, bone erosion and Rheumatology, Medical the histological evaluation of joint sections from animal University of Vienna, Vienna cartilage damage in these models. Unfortunately, 1090, Austria; models of inflammatory arthritis. The consensual published findings from many animal studies lack silvia. hayer@ meduniwien. ac. at approach of a task force including 16 academic and detailed description and appropriate assessment of industrial scientists as well as laboratory technicians the distinct histopathological features of arthritis: Received 6 October 2020 has resulted in the development of the Standardised methods of processing and scoring are poorly Revised 6 January 2021 Accepted 21 January 2021 Microscopic Arthritis Scoring of Histological sections defined, and often several histological variables (’SMASH’) recommendations for a standardised are combined into one score, thereby losing power processing and microscopic scoring of the characteristic to detect differences and making it impossible to histopathological features of arthritis, exemplified by four uncouple processes like joint inflammation and different rodent models for arthritis: murine collagen- destruction. While clinical trial designs are highly induced arthritis, collagen–antibody- induced arthritis, regulated,6–8 studies of experimental arthritis are http://ard.bmj.com/ human tumour necrosis factor transgenic Tg197 mice not standardised and every group may have its own and rat pristane- induced arthritis, applicable to any other methods for histological processing and scoring of inflammatory arthritis model. Through standardisation, joint sections, thereby hampering the combination the SMASH recommendations are designed to improve and comparison of multiple data sets. Therefore, and maximise the information derived from in vivo the European consortium BeTheCure, consisting arthritis experiments and to promote reproducibility and of 38 academic and industrial partners from 15 transparent reporting on such studies. In this manuscript, countries, funded by the Innovative Medicine on September 26, 2021 by guest. Protected copyright. we will discuss and provide recommendations for Initiative, a public–private partnership between the analysis of histological joint sections: identification of European Union and the European Federation of the regions of interest, sample preparation, staining Pharmaceutical Industries and Associations, set as procedures and quantitative scoring methods. In one of their main goals to standardise the histolog- conclusion, awareness of the different features of the ical evaluation of joint sections from animal models arthritis pathology in animal models of inflammatory of inflammatory arthritis. A task force team of 16 arthritis is of utmost importance for reliable research academic scientists, industrial scientists and labora- outcome, and the standardised histological processing tory technicians experienced in arthritis models has © Author(s) (or their and scoring methods in these SMASH recommendations therefore developed the Standardised Microscopic employer(s)) 2021. Re- use will help increase uniformity and reproducibility in Arthritis Scoring of Histological sections (‘SMASH’) permitted under CC BY. preclinical research on inflammatory arthritis. Published by BMJ. recommendations for a standardised processing and microscopic scoring of the histopathological To cite: Hayer S, features of arthritis, exemplified by four different Vervoordeldonk MJ, models, and applicable to any other inflammatory Denis MC, et al. Ann Rheum Dis Epub ahead INTRODUCTION arthritis model. Selected arthritis models included of print: [please include Day Inflammatory arthritides such as rheumatoid three of the most established systemic mouse Month Year]. doi:10.1136/ arthritis (RA) and psoriatic arthritis are common models, namely, collagen-induced arthritis (CIA), annrheumdis-2020-219247 systemic inflammatory diseases characterised by collagen–antibody-induced arthritis (CAIA) and Hayer S, et al. Ann Rheum Dis 2021;0:1–13. doi:10.1136/annrheumdis-2020-219247 1 Ann Rheum Dis: first published as 10.1136/annrheumdis-2020-219247 on 18 February 2021. Downloaded from Recommendation (>9/10). Representative images of joint sections were selected from a collection of histological sections from four animal models (CIA, CAIA, Tg197 and PIA) provided by the contrib- uting institutions of the task force members. RESULTS The BeTheCure task force defined 30 recommendations divided into seven categories for the standardisation of histological processing, evaluation, scoring and reporting of histopatho- logical features from inflammatory arthritis in mice and rats (table 1). Category 1: sample selection, orientation and regions of Figure 1 Common histopathological features of inflammatory joint interest for mouse and rat hind paw sections damage in human RA and experimental models. TRAP- stained joint Due to short reproduction times, relatively low costs, easy sections indicate synovitis, pannus formation, synovial osteoclast housing and handling and for ethical reasons, mice and rats formation, subchondral bone erosions as well as cartilage erosion in are the most commonly used species in animal experiments for human RA (upper row, MTP-1 joint section) and experimental arthritis arthritis research. Depending on the arthritis model, different models (lower row, affected tarsal joint from a 10- week- old Tg197 joints are affected and can be assessed for histopathological mouse). Original magnification is 50× (upper left), 100× (upper right; evaluation, including knee joints, carpal, tarsal and/or ankle lower left) and 200× (lower right). B, bone; BM, bone marrow; C, joints. Knee joints are only recommended for histopathological cartilage; P, synovial pannus; RA, rheumatoid arthritis; S, synovium; analysis in gonarthritis models like methylated bovine serum TRAP, tartrate- resistant acid phosphatase. albumin (mBSA)- mediated antigen- induced arthritis or after intra- articular injection of pathogenic mediators like cytokines and ligands like streptococcal cell wall (SCW) fragments into human tumour necrosis factor transgenic Tg197 mice as well as the knee joint. For systemic arthritis models affecting multiple the rat pristane- induced arthritis (PIA) model.9–16 Importantly, joints, such as CIA, CAIA, Tg197 and PIA, studying the histo- the standardised assessment of these four models allowed us to pathology of the hind paws is the most established and recom- highlight their differences regarding extent of synovial inflam- mended method. Clinical signs of arthritis such as joint swelling mation, subchondral bone erosion and articular cartilage damage and redness in hind paws or loss of grip strength can be non- or development of osteophytes, the latter not being untypical for invasively and longitudinally evaluated during the disease course RA but common in spondyloarthritis. These differences, as well and can be related to histological outcomes at the end of the as the different pathophysiological mechanisms like the relative study. Histological evaluation of affected front paws demon- contribution of the innate and/or the adaptive immune system strates limitations in standardisation due to lack of consistent to pathogenesis, determine which model suits the investigator’s cutting planes. research question best.3–5 17–19 Before starting an animal experiment, appropriate sample size calculation is essential for designing a scientifically conclu-